INFECTION AND IMMUNITY, OCt. 1990, p. 3202-3207 Vol. 58, No. 100019-9567/90/103202-06$02.00/0Copyright © 1990, American Society for Microbiology

Ingestion of Giardia lamblia Trophozoites by MurinePeyer's Patch MacrophagesDAVID R. HILL* AND RODERIKE POHL

Division of Infectious Diseases, Room L-3108, Department of Medicine, University ofConnecticut School of Medicine, Farmington, Connecticut 06030

Received 30 April 1990/Accepted 9 July 1990

Macrophages in Peyer's patches are important in the initiation of gastrointestinal immune responses toenteric pathogens. To examine their potential role in giardiasis, murine mononuclear cells were isolated fromcollagenase-treated Peyer's patches by their adherence to glass. These cells were incubated with Giardia lambliatrophozoites in the presence of nonimmune or immune (anti-Giardia antibody titer, .1:1,024) mouse serum.Macrophages ingested trophozoites at low levels when they were incubated with nonimmune mouse serum.Ingestion was significantly increased at all time points (P < 0.01) when cells and parasites were incubated in5% immune mouse serum; the number of Giardia trophozoites ingested per 100 macrophages was 21.6 ± 7.1(standard error of the mean) at 1 h and increased to 59.0 + 16.4 at 8 h. Electron microscopy documentedtrophozoite destruction within macrophages. Association of G. lamblia with macrophages elicited an oxidativeresponse; 50.9 ± 3.6% of macrophages with trophozoites attached or ingested reduced the dye Nitro BlueTetrazolium, compared with 13.0 ± 5.2% for cells without associated trophozoites (P < 0.04). These resultsdemonstrate that macrophages are capable of ingesting G. lamblia in vitro and may play an important role inhost defense in giardiasis.

The pathogenic protozoan Giardia lamblia is the mostfrequently isolated enteric parasite in the United States (4).Its importance as a pathogen has been recognized by itsassociation with symptomatic diarrhea throughout the world(10, 13), with chronic malabsorption in children (10, 42), andwith epidemic diarrhea after water-borne outbreaks (5, 24).Evidence supports a role for host immunity in clearance ofG. lamblia (17, 18, 36, 45), in protection from disease (22, 31,38), and, in certain instances, in production of disease,especially in individuals who demonstrate spruelike lesionson intestinal biopsy (7, 35). Experimental data in micesupport a role for both T and B lymphocytes in this immuneresponse. Athymic, nude mice are unable to clear theirinfection until they have been reconstituted with lympho-cytes (36). After reconstitution, it is likely that these miceclear their infection because they have been supplied withhelper-inducer T lymphocytes, which are required in theproduction of intestinal antibody (2, 3, 9, 17, 25). Intestinalanti-Giardia immunoglobulin G and secretory immunoglob-ulin A have been implicated both in protection against andclearance of G. lamblia (15, 45).Because G. lamblia is primarily a luminal parasite, the

gut-associated lymphoid tissue is likely to play the major rolein the immune response (1, 6, 18, 21, 43, 44). One of the firststeps in an effective gastrointestinal response is the ingestionand processing of antigen by macrophages residing in Pey-er's patches (1, 8, 21). Although macrophages from theperipheral blood and peritoneum have been demonstrated toingest G. lamblia (19, 23, 33), ingestion by Peyer's patchmacrophages (PPM4) has not been demonstrated in vitroand is the focus of this study. In transmission electronmicrographs of Peyer's patches from Giardia muris-infectedmice, Owen and colleagues demonstrated macrophagepseudopod penetration between columnar epithelial cellswith engulfment of trophozoites (30). In addition, there was

* Corresponding author.

lymphocyte rosetting around macrophages that had ingestedGiardia organisms, suggesting an antigen-processing func-tion for these cells. In previous work from our laboratory,human peripheral blood mononuclear phagocytes ingestedand destroyed G. lamblia trophozoites (19). Peritoneal mac-rophages from both rabbits and mice have also been shownto ingest Giardia organisms (23, 33). Ingestion in each casewas enhanced by the presence of anti-Giardia antibodies. Inthis study a mononuclear, adherent cell population consis-tent with macrophages was isolated from murine Peyer'spatches. These cells ingested G. lamblia trophozoites, andingestion was enhanced by anti-Giardia specific antibody.The interaction of macrophages with G. lamblia was asso-ciated with both an oxidative respiratory burst and withintracellular destruction of trophozoites.

(This work was presented in part at the annual meeting ofthe American Society of Tropical Medicine and Hygiene,Miami, Fla., November 1985.)

MATERIALS AND METHODSParasites. G. lamblia trophozoites, WB isolate (41) (30957;

American Type Culture Collection, Bethesda, Md.), wereaxenically cultivated in filter-sterilized Diamond modifiedTrypticase (BBL Microbiology Systems, Cockeysville, Md.)Panmede (Paines and Byrne Ltd., Greenford, England)serum medium (46) as previously described (20). Ten percent(vol/vol) heat-inactivated (AHI; 56°C for 30 min) fetal calfserum (FCS; GIBCO Laboratories, Grand Island, N.Y.)replaced horse serum, and 3% (vol/vol) NCTC vitaminmixture no. 108 (GIBCO) was used. After 2 to 5 days ofculture, G. lamblia trophozoites were harvested by coolingculture tubes to 4°C and then sedimenting the parasites at250 x g for 5 min. Trophozoites were washed four times inDulbecco phosphate-buffered saline (pH 7.4) and suspendedin RPMI 1640 for the macrophage ingestion assay.

Peyer's patch macrophages and mouse sera. The isolationof murine PPMX was adapted from previously establishedmethods (28, 34) that utilize enzymatic digestion of Peyer's

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patches and differential adherence to enrich the populationfor macrophages. Eight-week-old female BALB/c mice (spe-cific pathogen free; Portage, Mich., Charles River BreedingLaboratories) were screened for intestinal parasites beforeuse (37) and have been consistently negative over severalyears. Mice were anesthetized with ether and sacrificed bycervical dislocation. With sterile technique the abdomenswere opened, and the intestines were removed and flushedwith cold Hanks balanced salt solution with Ca2" and Mg2+.Peyer's patches were identified, removed, and placed in coldRPMI 1640 with 5% (vol/vol) FCS, penicillin (100 U/ml), andgentamicin (50 ,g/ml). They were teased apart, the debriswas allowed to settle, and the supernatant fluid containingcells was washed twice with fresh medium. The debris wasthen treated with 200 U of collagenase (type IA; SigmaChemical Co., St. Louis, Mo.) per ml at 37°C for 60 min todissociate adherent cells, increasing the yield of PPM4 (11,28, 34, 48). Cells in the supernatant were washed three timesand combined with the previous fraction. All cells wereenumerated by counting in a hemacytometer, tested forviability by trypan blue (0.1%) exclusion, and differentiatedby May-Grunwald and nonspecific esterase staining (49) ofcytocentrifuge preparations. By this method 2.9 x 107 + 0.9x 107 (standard deviation) cells per mouse were obtainedwith greater than 90% viability. On cytocentrifuge prepara-tions 7.5 + 2.7% (standard deviation) of the cells werenonspecific esterase stain positive, indicating that they weremacrophages (49).Washed supernatant cells were concentrated to 107/ml,

and 0.5-ml samples were placed on 22-mm2 glass cover slipsand incubated at 37°C in 5% C02-95% air. After 2 h,nonadherent cells were gently washed off, and the cover slippreparations were flooded with fresh medium containing15% FCS and antibiotics. This procedure yielded approxi-mately 1 x 103 to 5 x 103 mononuclear adherent cells percover slip.Serum was obtained by tail bleeds from control, nonim-

munized mice and mice immunized with whole G. lambliatrophozoites. Trophozoites were washed in phosphate-buff-ered saline and then mixed 1:1 with complete Freund adju-vant (Sigma). Then 106 trophozoites in 200 RI were injectedinto the peritoneal cavity and hind footpad of each mouse,followed by a second injection after 4 weeks. Mice were bled4 weeks after this. Serum anti-G. lamblia antibody titersfrom immunized mice, determined by an immunofluores-cence assay (47) with a polyclonal goat anti-mouse antibody(Organon Teknika, West Chester, Pa.), were .1:1,024. Theanti-G. lamblia antibody titer from control mice was s1:4.Macrophage ingestion assay. G. lamblia trophozoites (5 x

105) were added to duplicate PPM4 cover slip preparationsin RPMI 1640 with 15% AHI FCS alone, 15% FCS plus 5%AHI nonimmune mouse serum (NIMS), or 15% FCS plus 5%AHI immune mouse serum (IMS). To measure the ability ofthe PPM4) to ingest particles, zymosan (0.5 mg/ml) that hadbeen opsonized with complement (10% fresh mouse serum,30 min, 37°C) was added to separate control cover slips.

After 1, 2, 4, and 8 h, acridine orange (5 ,ug/ml) was addedto cover slip preparations to stain macrophage lysosomesand to aid in the distinction of intracellular from extracellularG. lamblia (19, 32). Only cells that were adherent, mononu-clear, and with abundant lysosomes were examined for theirinteraction with G. lamblia. Trophozoites that had beeningested by macrophages were enumerated by phase-con-trast and fluorescence microscopy with an Olympus BHTmicroscope fitted with reflected light fluorescence. At least

100 cells per cover slip from multiple fields were counted;five separate experiments were carried out.

Electron microscopy. PPM4 cover slip preparations thathad been incubated with trophozoites for 2 h were cooled for2 h at 4°C. Cells were gently removed with a rubberpoliceman and pelleted. The supernatant was removed, andthe pellet was fixed with 4% glutaraldehyde in cacodylatebuffer for 1 h, followed by 2% buffered osmium tetroxide for30 min. After dehydration in a graded series of alcohol andpropylene oxide washes, specimens were embedded in epon-araldite. After polymerization, 60-,um sections were cut,stained with uranyl acetate and lead citrate, and examinedwith a Philips EM 300 electron microscope.

Assay of oxidative response. An oxidative response of thePPM4s was measured qualitatively by reduction of the dyeNitro Blue Tetrazolium (NBT; Sigma) (12). PPM4 cover slippreparations were incubated with G. lamblia trophozoitesfor 2 h or opsonized zymosan for 1 h. Preparations were thenincubated with NBT (1.5 x 10-3 M) for 20 min, washed, andcounterstained with safranin. The percentage of mononu-clear cells that contained aggregated formazan deposits ofblue was determined by light microscopy. Each conditionwas studied with duplicate cover slip preparations fromthree separate experiments.

Statistical analysis. Comparisons between groups wereevaluated with a paired t test or Student's two-tailed t testfor independent means.

RESULTSIngestion of G. lamblia by PPM+. PPM+s ingested G.

lamblia trophozoites at low levels when they were incubatedwith FCS alone or NIMS and readily ingested them when

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FIG. 1. Ingestion of Giardia trophozoites by PPM+s. PPM4sincubated with trophozoites in NIMS (Ei) or FCS alone (L) werecapable of ingesting trophozoites at low levels. Ingestion wassignificantly increased at each time point (P < 0.01) when cells andparasites were incubated in 5% IMS (-). PPM+ cover slip prepara-tions incubated with G. lamblia trophozoites were observed underphase-contrast microscopy at 1, 2, 4, and 8 h. Each point representsthe mean of five separate experiments. Bars represent standarderrors.

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FIG. 2. Transmission electron micrograph of a macrophage with an ingested Giardia trophozoite (GL). Parasite flagellae are readily seen(arrow); one Giardia trophozoite is extracellular (gl). Macrophages were incubated with G. lamblia for 2 h at 37°C with 5% AHI IMS and thenprocessed for electron microscopy. N, Macrophage nucleus. Bar, 1 p.m.

incubated with 5% AHI IMS (Fig. 1). Ingestion was con-firmed by electron microscopy (Fig. 2). At each of the fourtime points, ingestion by PPMX of parasites incubated withIMS was significantly greater (P < 0.01) than ingestion withFCS alone or NIMS. The number of trophozoites ingested inimmune serum per 100 PPM+s increased from 21.6 ± 3.6(standard error of the mean) at 1 h to 59.0 ± 16.4 at 8 h. Over70% of maximum ingestion occurred by 2 h. On controlcover slips, 72.7 ± 3.5% (standard error of the mean) of theadherent cells ingested zymosan; a maximum of 33.4 ± 3.4%ingested G. lamblia.

Survival of G. lamblia trophozoites incubated at 37°C inRPMI 1640 and 15% FCS with 5% AHI IMS in 5% C02-95%air was excellent as determined by strict morphologic crite-ria, a correlate of survival by growth (20). At 1 h, 95.4 +

1.2% (standard error of the mean) were alive, 92.5 + 1.1%were alive at 2 h, 89.2 ± 1.0% were alive at 4 h, and 87.1 +

2.4% were alive at 8 h (four experiments). Trophozoitemovement and flagellar motility frequently could be docu-mented when parasites were within macrophages, indicatingthat macrophages were binding to and ingesting viableparasites.The destruction of trophozoites within macrophages was

illustrated by electron microscopy. In Fig. 3 trophozoiteswith extensive disintegration can be demonstrated, indicat-ing trophozoite death.

Oxidative response. Macrophage interaction with G. lam-

blia was associated with an oxidative response as measuredby NBT reduction. Table 1 indicates that 50.9 + 3.6%(standard error of the mean) of macrophages that hadingested G. lamblia or had trophozoites attached to themwere NBT positive, compared with 13.0 + 5.2% of cellswithout associated trophozoites (P < 0.04). NBT reductionby cells associated with trophozoites was also significantlygreater than NBT reduction by macrophages on controlcover slips incubated in 5% IMS alone (P < 0.04). Thepercentage of cells with zymosan or G. lamblia associatedwith them was similar to that seen in the ingestion assay,72.3 ± 7.2 or 22.9 ± 9.7%, respectively.

DISCUSSION

In this study a mononuclear cell population was isolatedfrom Peyer's patches and studied in its interaction withGiardia trophozoites. These cells were adherent, nonspecificesterase stain positive, phagocytic, and oxidatively active,characteristics consistent with their being macrophages.They ingested Giardia trophozoites, as documented byphase-contrast microscopy and confirmed by electron mi-croscopy. Ingestion of parasites was significantly enhancedby the addition of anti-Giardia specific antibody, consistentwith the findings from previous work investigating macro-phages and Giardia species (19, 23, 33). Some Giardiatrophozoites that were ingested demonstrated morphologic

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FIG. 3. Transmission electron micrograph of a macrophage containing degenerated trophozoites (arrows), indicating parasite death. Thereis extensive disintegration, with only trophozoite disks clearly recognizable (arrows). An intact Giardia trophozoite (GL) can be seen adheringto the macrophage. Bar, 1 R,m.

destruction, indicating killing by macrophages. The ability ofthese cells to ingest G. lamblia supports an expected role forthem in the gastrointestinal immune response.Both as a measure of the functional capacity of the PPMX

and as a potential microbicidal mechanism, the oxidativeactivity of the cells was examined. Association of G. lambliawith macrophages produced an oxidative burst, as measuredby NBT reduction. We have previously demonstrated thatGiardia organisms are susceptible to one product of theoxidative burst, H202, at a concentration of .5 x 10' M

TABLE 1. Reduction of NBT by PPM4sa

% of PPM4 (mean + SEM)PPM4 incubation

condition NBT false-NBT positive positiveb

PPM4 + G. lamblia + 5% IMS 50.9 + 3.6c 13.0 ± 5.2PPM4 + zymosan 64.2 ± 115.d 4.9 ± 1.5PPMf + 5% IMS alone 14.9 ± 1.8

a Data are three experiments, each done in duplicate.b Percent of cells on the same cover slip that were NBT positive but did not

have either G. lamblia or zymosan associated with them.Pp < 0.04 compared with either the percent false-positive NBT for the cellson the same cover slip, or the percent positive NBT for PPM4 incubated with5% IMS alone.

d p < 0.03 compared with percent false-positive NBT.

(19). The addition of lactoperoxidase and KI, found inimmature macrophages and monocytes, enhanced killing byH202. Another potential mechanism of parasite killing bymacrophages was fusion of phagosomes with lysosomes,which was observed on phase-contrast microscopy of theacridine orange-stained preparations (data not shown).There is little information on gastrointestinal macro-

phages. Recent investigations with human and animal intes-tine have demonstrated the need to digest tissue enzymati-cally before adherent macrophages are released (11, 14, 28,34, 48). Once tissue has been digested, small numbers ofmacrophages ranging from 0.2 to 10% of the cells areavailable for study. In our study, 8% of the collagenase-treated Peyer's patch cells were macrophages, as deter-mined by nonspecific esterase staining (49). The cells thathave been isolated have demonstrated characteristics con-sistent with macrophages, such as adherence, monoclonalantibody binding, surface Fc receptors, nonspecific esterasestaining, and the ability to ingest particles and to function asaccessory cells in antigen-specific lymphocyte transforma-tion (11, 14, 26, 28, 29, 34, 48). The current investigation nowdocuments the ability of PPM+s to ingest Giardia trophozo-ites in vitro. In conjunction with those that have examinedhuman peripheral and animal peritoneal macrophages (19,23, 33, 40), this study expands a potential role for macro-phages in giardiasis.

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3206 HILL AND POHL

The PPMX is of particular interest because of its impor-tance as an antigen-presenting cell in the development of animmune response to enteric antigens (8, 21, 26). This role ingiardiasis was suggested by electron microscopy of Peyer'spatches from G. muris-infected mice, in which lymphocyteswere seen in a rosette around macrophages with ingestedGiardia trophozoites (30). Because of the small numbers ofmacrophages that reside in the mucosa, lumen, or Peyer'spatches of the intestine (11, 16, 27, 28, 34, 48) and theinfrequent documentation of penetration of Giardia organ-isms beyond the intestinal epithelium (30, 39), these cellsmay function less as effector cells. However, the ability toingest Giardia trophozoites supports this potential role as aneffector cell and allows them to participate in antigen proc-essing of Giardia organisms. Further study in this area willhelp to define the multiple important factors in host immu-nity in giardiasis.

ACKNOWLEDGMENTS

This work was supported in part by a grant from the University ofConnecticut Research Foundation and by Public Health Servicegrant AI-22438 from the National Institute of Allergy and InfectiousDiseases. D. R. Hill is a recipient of the Smith, Kline and FrenchLaboratories Young Investigator Award of the Infectious DiseasesSociety of America.We thank Connie Gillies of the Clinical Electron Microscope

Facility at the University of Connecticut Health Center for assis-tance in obtaining the electron micrographs and Edward Pesanti forhelpful comments and suggestions.

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